Influenza is an enveloped RNA virus of the orthomyxovirus family and a Category C biodefense pathogen of interest to the NIAID. Despite significant advances in the development of both vaccines and drugs, influenza viruses infect 5-15% of the human population every year, resulting in 3-5 million cases of severe illness and 250,000-500,000 deaths annually. New strains of influenza that are resistant to vaccines emerge every year, and existing drugs against influenza are often easily evaded. New drugs against influenza are needed that inhibit diverse strains of the virus and that resist viral evasion. This proposal will result in a novel assay for the discovery of therapeutics against influenza. Two of the four FDA-approved therapeutics against influenza target the M2 ion channel, making M2 a validated molecular target for the treatment of influenza infection. However, developing new therapeutics that target M2 has been difficult. Ion channels have been troublesome targets for traditional high-throughput screening (HTS), in large part because cell-based ion channel assays restrict the application of preferred HTS strategies. New methods of manipulating ion channels and detecting their activity are needed to aid the development of new drugs against difficult ion channel targets such as M2. The goal of this proposal is to develop a high-throughput screening assay that can identify novel inhibitors against influenza M2 proteins. Also of significance to the NIAID, the methods and techniques that result from this proposal will also be useful for the discovery of new viral ion channels, new inhibitors of ion channels within other viruses, and research into understanding the contribution of viral ion channels to viral replication and pathology. The Specific Aims of this proposal are to: [unreadable] I. Establish a Primary HTS Assay for Compounds that Block M2 Activity. [unreadable] II. Test M2 Activity Assay in Proof-of-concept HTS. [unreadable] [unreadable] [unreadable]